Liquid chromatographic techniques are well known in the separation of chemical or biological compounds. Chromatographic separations of biological materials may be accomplished through the use of high performance (or high pressure) liquid chromatography (HPLC), and reverse phase HPLC has been found to be particularly useful. Chromatographic packings are made from small, porous particles with suitable, organic moieties on the surface. They are loaded into a hollow column, and the sample containing the materials to be separated is introduced into the column and allowed to flow through, either by the pull of gravity or by mechanical action, as for example through the use of an associated pump.
Once such biological or chemical materials have been separated into zones, they must be identified. In many instances the compounds must also be quantified. To this end, various techniques have been developed. These include the ultraviolet absorption profile of the column eluate. Associated chart recorders or other devices indicate by a change in the transmission of ultraviolet light through the flow path when detectable species enter the detector. Such a detection system requires a sufficient quantity of materials to provide detectable amounts. In many instances, the small quantities of available samples, difficulties in resolving molecular species of similar size and charge, as well as the presence of large amounts of sample matrix materials with similar bulk properties preclude effective analysis using the general detection scheme indicated above.
Many samples for which detection and identification is desirable are of compounds present in amounts too small to be quantified by conventional procedures. Thus, many other techniques such as those generally characterized by antibody-antigen reactions have been developed.
An immunogen is a chemical or biological substance capable of eliciting production of antibodies when it is introduced into a responding organism. An antigen is a substance that is bound by the antibody. The challenged organism responds via the immunological production of antibodies directed specifically towards the foreign antigen. Once isolated and purified, antibodies capable of binding to a specific given antigen become very useful laboratory reagents. Such antibodies may be tagged with radioactive or fluorescent labels, and an antibody-antigen complex may be formed in a reaction vessel when a given antigen is exposed to such antibodies. The antigen-antibody complexes may be separated from the reaction vessels and indirectly quantified on the basis of the label present in the complex. Enzymes may also be similarly linked to appropriate antibodies and the presence of the enzyme-antibody-antigen complex may be detected by measuring the reaction of the enzyme with a given substrate.
Alternatively, such labels may be coupled to antigen molecules, thereby giving rise to competitive binding assays to determine the presence and quantity of a given antigen based upon the competition for antibody binding sits between a known amount of labeled antigen and an unknown amount of unlabeled antigen present in a particular chemical or biological sample material. Such techniques are well known in the art.
It is also known in the art to attach antibodies to a solid support, thereby forming a testing material that can specifically bind to labeled or unlabeled antigens in a chemical or biological sample, which presence may be subsequently detected by the monitoring of fluorescent or radioactive labels, for example. These types of assays are generally discussed in U.S. Pat. Nos. 4,273,756, 4,272,505, 4,272,504, and 4,277,560.
The '560 patent discloses the use of immobilized antibodies that are saturated with enzyme-antigen complexes to reversibly bind to the immobilized antibody. The unlabeled antigen in a given sample is injected into a stream that flows directly into a first stage packed column containing the immobilized antibody, and a competitive equilibrium process takes place between the antigen-enzyme complex bound to the antibody and the unlabeled antigen of the sample as it flows past the antibodies immobilized on the substrate. Thus, a given quantity of labeled antigen is released into the flowing stream, to be measured downstream after reaction with a second, or detection, stage, such as a suitable enzyme substrate. A measurable reaction product may be quantitatively determined, and the concentration of the unlabeled antigen in the original sample may also be determined, such concentration being related to the amount of enzyme activity detect by the second stage. The '560 patent teaches the use of a first stage device having antibodies specific for only a single antigen. Thus, this technique is useful only for the quantification of antigens which have previously been identified, and a different immobilized antibody stage would be required for the quantification of a second antigen.
The '505 patent discloses a thyroid hormone assay involving competitive binding of labeled antigens with the same antigen from a biological fluid sample. The amount of thyroid hormone in the sample may be calculated from the fluorescence measurement of the sample as compared with a standardized data table from the assay.
No related method or apparatus is known that allows the convenient quantification of a plurality of antigens from a given biological sample using a post-chromatographic separation antibody-antigen competitive binding component.